Assay for Detection of Transient Intracellular CA2+

ABSTRACT

This invention relates to a simple end point assay for detection of transient intracellular Ca 2+  with broad applicability to many Ca 2+ channel proteins comprising, Generation of expression constructs for the fusion proteins having the Ca 2+/ calmodulin dependent protein kinase II (CaMKII) phosphorylation sites of NR2A or NR2B subunits of N-methyl-D-aspartate receptor (NMDAR) or the voltage gated potassium channel of  Drosophila  (Eag) or any protein sequence which binds to the T-site of CaMKII similar to NR2B, conjugated to mitochondrial localizing signal sequence, or mutants of these sequences as described herein. Generation of mammalian expression constructs of α-CaMKll as a chimera with green fluorescent protein (GFP-α-CaMKII) or its mutants as described herein. Site-Directed mutagenesis, Transfection, Ca 2+  stimulation, imaging and quantification of the number of cells with Ca 2+ -dependent signal, wherein, NMDA receptor activity assay, TRPVI receptor activity assay, GluR4 receptor activity assay are performed to detect the activity Of Ca 2+  channel proteins.

FIELD OF INVENTION

The invention relates to detection and quantification of transientchanges in intracellular metabolites by making use of a resultingprotein-protein interaction that is irreversible and longlasting.

BACKGROUND OF INVENTION

Increase in concentration of intracellular free Ca²⁺ ions is a widelyused signaling mechanism under various physiological conditions. Releaseof free Ca²⁺ ions inside the cell happens either through plasma membranechannel proteins that allow Ca²⁺ influx or by release from intracellularCa²⁺ stores. Since Ca²⁺ release thus becomes the activity of a widevariety of proteins, methods for detection and measurement ofintracellular free Ca²⁺ are used as assays for many proteins that arepotential drug targets. These methods should be capable of sensing thetransient rise in intracellular [Ca²⁺] and also of generating adetectable signal.

Most of the commonly used methods for Ca²⁺ sensing use molecules thatexhibit changes in their fluorescence characteristics upon exposure toincrease in [Ca²⁺]. These include small molecular weight macromoleculessuch as fura-2, indo-1, fluo-3, and Calcium-Green (U.S. Pat. Nos.4,603,209 and 5,049,673) as well as proteins such as GFP (greenfluorescent protein) family of proteins with a sensor peptide insertedwithin them (U.S. Pat. No. 6,469,154) or the cameleon molecules oftandem GFP constructs with an intervening Ca²⁺ sensing peptide sequence,that generate a FRET signal upon sensing free Ca²⁺ (U.S. Pat. No.5,998,204). In all these methods, the fluorescence signal is also asshort-lived as the Ca²⁺-transient, and hence signal acquisition inreal-time is mandatory. Moreover this also imposes rapid liquiddispensing. These factors not only necessitates the use of moreexpensive equipments but also limits the choice of equipments that canbe used in addition to imposing severe constraints for carrying outthese assays at high throughput scale. U.S. Pat. No. 6,514,709 discloseda method that prolongs the duration of the Ca²⁺-transients byincorporating an intracellular chelator that alters the kinetics ofsignal generation, so that the signal is prolonged. However in all thesemethods, measurements will have to be done on cells in the livecondition.

OBJECTS OF THE INVENTION

The main object of this invention is to detect intracellular Ca²⁺transients using simple end point assay.

The other object is to develop a Ca²⁺ sensing assay with broadapplicability to many Ca²⁺ channel proteins.

Another object is to detect intracellular Ca²⁺ transients using a simplemethod wherein the need for realtime measurements on live cells isavoided.

STATEMENT OF INVENTION

This invention relates to a simple end point assay for detection oftransient intracellular Ca²⁺ with broad applicability to manyCa²⁺channel proteins comprising, Generation of expression constructs forthe fusion proteins having the Ca²+/calmodulin dependent protein kinaseII (CaMKII) phosphorylation sites of NR2A or NR2B subunits ofN-methyl-D-aspartate receptor (NMDAR) or the voltage gated potassiumchannel of Drosophila (Eag) or any protein sequence which binds to theT-site of CaMKII similar to NR2B, conjugated to mitochondrial localizingsignal sequence, or mutants of these sequences as described herein.Generation of mammalian expression constructs of α-CaMKll as a chimerawith green fluorescent protein (GFP-□-CaMKII) or its mutants asdescribed herein. Site-Directed mutagenesis, Transfection, Ca²+stimulation, imaging and quantification of the number of cells withCa²⁺-dependent signal, wherein, NMDA receptor activity assay, TRPVIreceptor activity assay, GluR4 receptor activity assay are performed todetect the activity of Ca²+ channel proteins.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 is a schematic representation of the assay system for detectionof release of intracellular free Ca²⁺.

FIG. 2 shows Ca²⁺-dependent generation of signal due to GFP-α-CaMKIItranslocation towards mitochondrially localized NR2B sequence (MLS-NR2B)observed as the appearance of punctae.

FIG. 3 shows Ca²⁺-dependent generation of signal due to GFP-α-CaMKIItranslocation towards full-length NR2B localized on intracellularmembranous organelles.

FIG. 4 shows two images of cells, one obtained after inducingCa²⁺-influx to cells (Image 1) and the other obtained without inducingany Ca²⁺-influx (Image 2).

FIG. 5 shows the qunatitation data obtained as described in FIG. 4, forthe wild-type and T286D mutant forms of GFP-α-CaMKII.

FIG. 6 shows the quantitation data obtained as described in FIG. 4, forthe wild-type and T286D mutant forms of GFP-α-CaMKII, at differentconcentrations of Ca²⁺ in the extracellular medium.

FIG. 7 shows Ca²⁺-dependent generation of signal due to GFP-α-CaMKIItranslocation towards mitochondrially localized Eag sequence (MLS-Eag)

FIG. 8 shows signal generated due to Ca²⁺-influx through NMDA receptorformed of NR1 and NR2B subunits.

FIG. 9 shows signal generated due to Ca²⁺-influx through NMDA receptorformed of NR1 and NR2A subunits.

FIG. 10 shows signal generated due to Ca²⁺-influx through vanilloidReceptor (TRPV1)

FIG. 11 shows signal generated due to Ca²⁺-influx through the AMPA-typeglutamate receptor, GluR4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an assay for detecting intracellularbinding between two proteins. The assay is useful for detectingintracellular release of free Ca²⁺. In this assay method the transientCa²⁺ signal is captured by an irreversible protein-protein interactionso that the signal is converted into a form that can be preserved fordays to even months. There is absolutely no requirement of any real timesignal acquisition or live cell signal acquisition.

Increase in concentration of intracellular free Ca²⁺ ions is a widelyused signaling mechanism under various physiological conditions. Releaseof free Ca²⁺ ions inside the cell happens either through plasma membranechannel proteins that allow Ca²⁺ influx or by release from intracellularCa²⁺ stores. Since Ca²⁺ release thus becomes the activity of a widevariety of proteins, methods for detection and measurement ofintracellular free Ca²⁺ are used as assays for many proteins that arepotential drug targets. These methods should be capable of sensing thetransient rise in intracellular [Ca²⁺] and also of generating adetectable signal.

Most of the commonly used methods for Ca²⁺ sensing use molecules thatexhibit changes in their fluorescence characteristics upon exposure toincrease in [Ca²⁺]. These include small molecular weight macromoleculessuch as fura-2, indo-1, fluo-3, and Calcium-Green (U.S. Pat. Nos.4,603,209 and 5,049,673) as well as proteins such as GFP (greenfluorescent protein) family of proteins with a sensor peptide insertedwithin them (U.S. Pat. No. 6,469,154) or the cameleon molecules oftandem GFP constructs with an intervening Ca²⁺ sensing peptide sequence,that generate a FRET signal upon sensing free Ca²⁺ (U.S. Pat. No.5,998,204). In all these existing methods, the fluorescence signal isalso as short-lived as the Ca²⁺-transient, and hence signal acquisitionin real-time is mandatory. This requires sophisticated equipments withincreased temporal resolution and capability for real time measurementsfrom live cells. Moreover this also imposes rapid liquid dispensing.Consequently the cost of instrumentation becomes significantly high aswell as the choice of equipment§ that can be used becomes limited. Inaddition severe constraints are imposed for carrying out these assays athigh throughput scale. U.S. Pat. No. 6,514,709 disclosed a method thatprolongs the duration of the Ca²⁺-transients by incorporating anintracellular chelator that alters the kinetics of signal generation, sothat the signal is prolonged. Even then, measurements will have to bedone on cells in the live condition.

Ca²⁺/calmodulin dependent protein kinase type II (CaMKII) present atneuronal synapses, upon activation by increase in intracellular Ca²⁺,gets translocated to the NMDA-type glutamate receptor and bindsirreversibly by its T-site (the Thr²⁸⁶-binding site) to the NR2B subunitof the receptor. A GFP-fusion of CaMKII (GFP-CaMKII) and a fusion of thebinding sequence on NR2B with a mitochondrial targeting sequence(MLS-NR2B) upon co-transfection in HEK-293 cells, bind to each other inresponse to increase in intracellular Ca²⁺. GFP-CaMKII present in thecytosol translocates to mitochondria, where it binds to MLS-NR2B. FIG. 1depicts schematically the appearance of punctae of green fluorescence incells which had a uniform distribution of green fluorescence beforestimulation with Ca²⁺. To demonstrate this phenomenon, intracellularCa²⁺ influx was generated by adding the ionophore, ionomycin, whichbinds to the cell membrane and forms pores through which extracellularCa²⁺ flows in. This is a combination of two published methods (Strack,S., McNeill, R. B., and Colbran, R. J., 2000, J Biol Chem 275,23798-23806; Bayer, K. U., LeBel, E., McDonald, G. L., O'Leary, H.,Schulman, H. and De Koninck, P., 2006, J. Neurosci. Vol. 26, p1164-1174) but is not identical to either.

Surprisingly it has been found that the percentage of green cells thatformed green punctae increases with increase in concentration ofextracellular Ca²⁺ provided. At higher concentrations of extracellularCa²⁺, more cells formed green punctae. Since extracellular concentrationof Ca²⁺ is related to the

EXPERIMENTAL PROCEDURES

Generation of Expression Constructs for the Fusion Proteins having thePhosphorylation Sites on NR2A or NR2B, Conjugated to MitochondrialLocalizing Signal Sequence

The cDNAs of NR2A (encoding amino acid residues 1265-1301) or NR2B(encoding amino acid residues 1271-1311) harboring the phosphorylationsite for CaMKII (Ser¹²⁹¹ and Ser¹³⁰³ of NR2A and NR2B respectively)present between the BamHI and EcoRI sites in pGEX-2T-NR2A orpGEX-2T-NR2B vectors (Praseeda, M., Mayadevi, M., and Omkumar, R. V.,2004, Biochem. Biophys. Res. Commun. 313, 845-849) were subcloned intothe pSGH vector. The sequences of NR2A and NR2B form parts of fusionproteins with a mitochondrial targeting signal sequence, a myc-epitopeand GST at the N-terminal end (MLS-NR2A or MLS-NR2B). The sequences ofthe fusion constructs were confirmed by DNA sequencing of the NR2A orNR2B segments.

Generation of Mammalian Expression Constructs of α-CaMKII

The cDNA of α-CaMKII was amplified by PCR from pGEM2α, (Praseeda M.,Pradeep K. K., Krupa A., Krishna S. S., Leena S., Kumar R. R., CheriyanJ., Mayadevi M., Srinivasan N. and Omkumar R. V., 2004, Biochem J, 378,391-397) using forward primer having nucleotide sequence as set forth inSEQ ID NO: 1 and reverse primer having nucleotide sequence as set forthe in SEQ ID NO: 2 to yield a PCR product with KpnI and BamHI sites at5′ and 3′ ends respectively. The PCR product was cloned into pEGFP-C1vector (Clontech) using the corresponding restriction sites in themultiple cloning site of the vector, which resulted in an N-terminalGFP-fusion of α-CaMKII (GFP-α-CaMKII).

5′-CGGGGTACCATGGCTACCATCACCTGC-3′ SEQ ID NO: 15′-CGCGGATCCCTATCAATGGGGCAGGACGG-3′ SEQ ID NO: 2

Site-Directed Mutagenesis

Site-directed mutagenesis was carried out using the QuikChangeSite-Directed Mutagenesis Kit from Stratagene based on Kunkel's method(Kunkel, T. A., 1985, Proc. Natl. Acad. Sci. USA 82, 488-492). The cDNAof α-CaMKII present in the expression vector pEGFP-C1-α-CaMKII served asthe template to generate mutants. The NR2A or NR2B sequences present inthe pSGH-NR2A or pSGH-NR2B expression vectors were also used astemplates to generate mutants.

Transfection, Ca²⁺-Stimulation and Imaging

HEK-293 (Human Embryonic Kidney) cells were cultured as monolayer inDMEM containing 10% FBS and antibiotics (penicillin 100 units/ml,streptomycin 100 μg/ml, fungizone 2.5 μg/ml) at 37° C. in a humidatmosphere having 5% CO₂.

For transfection, 1.5×10⁴ HEK-293 cells were seeded on sterile 12 mmcover slips in 24 well plates (Nunc). After 24 hrs, the cells werecotransfected with plasmid constructs encoding GFP-α-CaMKII (0.1μg/well) and either of MLS-NR2A or MLS-NR2B (0.25 μg/well), or pRK-NR2Aor pRK-NR2B (along with pRc-NR1 for expression of functional NMDAreceptor) using Lipofectamine reagent. About 48 hrs followingtransfection, cells were washed with Hank's balanced salt solution(HBSS) containing 20 mM HEPES and 1.4 mM EGTA. The cells were thenincubated with 15 μM ionomycin in the same buffer for 10 min.Subsequently the cells were incubated in the same buffer containing 2 mMCaCl₂ and 3 μM ionomycin. Cells were fixed with 3.7% paraformaldehyde inPBS (Phosphate-buffered saline, 10 mM disodium hydrogen phosphate, 1.8mM potassium dihydrogen orthophosphate, 140 mM sodium chloride, 2.7 mMpotassium chloride, pH adjusted to 7.4) at different time points afteraddition of calcium, mounted with mountant containing DABCO (antifade)and were observed through either a simple fluorescence microscope or aconfocal fluorescence microscope (TCS-SP2-AOBS from Leica).

Appearance of perinuclear granules due to mitochondrial localization ofGFP-α-CaMKII is taken as the signal for binding between GFP-α-CaMKII andMLS-NR2B. In case of binding between GFP-α-CaMKII and full-length NR2B,accumulation of GFP-fluorescence in the membraneous structures such asnuclear membrane, endoplasmic reticulum membrane, plasma membrane, etcis taken as signal for binding. Data presented are for cells fixedimmediately after incubation with 15 μM ionomycin (−Ca²⁺ influx) or 2-5minutes after Ca²⁺ stimulus (+Ca²⁺ influx). The total number of greenfluorescent cells and cells which develop the signal for binding werecounted manually for each microscopic field. Number of cells thatdevelop signal for binding is represented as percentage of total numberof green fluorescent cells (number of green fluorescent cells whichdevelop signal for binding+number of green fluorescent cells which donot develop signal) in a field. Average for at least three separatefields was obtained for each experiment. Mean±standard deviation of dataobtained from three such experiments is presented as values in inset inFIG. 2. Wherever images for lack of binding are presented, no cell wasfound to develop the perinuclear granules in repeated experiments andhence no quantitation data is presented.

NMDA Receptor Activity Assay

For expression of NMDA receptor (NMDAR), the cells were cotransfectedwith plasmid constructs encoding GFP-α-CaMKII (0.1 μg/well) and NR1(pRc-NR1 plasmid) and NR2B (pRK-NR2B plasmid) subunits of NMDAR (0.33μg/well of pRc-NR1 and pRK-NR2B) using Lipofectamine (Invitrogen). Whengrowth medium was added to the cells, it was supplemented with aninhibitor of NMDA receptor, either 1 mM of 2-amino-5-phosphovaleric acid(AP-5) or 10 μM MK-801. For detecting activity of NMDAR, about 48 hrsfollowing transfection, cells were washed twice with Ca²⁺ and Mg²⁺ freeHank's balanced salt solution (HBSS). The cells were then incubated withHBSS solution containing glutamate (10 μM) and glycine (2 μM), with orwithout CaCl₂ (2 mM) for about 5 minutes. Cells were fixed with 3.7%paraformaldehyde in PBS for about 10 to 15 minutes and were washedthrice with PBS. The fixed cells were mounted onto glass slides.

In experiments with NMDAR containing NR2A subunit, cells wereco-transfected with plasmid constructs encoding GFP-α-CaMKII (0.1μg/well) and NR1 (pRc-NR1 plasmid) and NR2A (pRK-NR2A plasmid) subunitsof NMDAR (0.33 μg/well of pRc-NR1 and pRK-NR2A) along with MLS-NR2B(0.33 μg/well of pSGH-NR2B) and growth medium was not supplemented withAP-5. NMDAR activity was detected as described in the previous section.

TRPV1 Receptor Activity Assay

For the expression of TRPV1, HEK-293 cells were cotransfected with TRPV1(pCDNA3-TRPV1, about 0.2 μg/well), MLS-NR2B (pSGH-MLS-NR2B, about 0.33μg/well) and GFP-CaMKII (pEGFP-CaMKII, about 0.1 μg/well) usinglipofectamine reagent. After 48 hours, the transfected cells weresubjected to TRPV1 activity assay. The growth medium was removed and thecells were washed with Ca²⁺ and Mg free HBSS containing 400 μM EGTA. Thereceptor was activated with 3 μM or 10 nM of capsaicin with 2 mM Ca²⁺ inHBSS without Mg²⁺ for 5 minutes. Control without Ca²⁺ during activationwas also performed. The inhibitor of TRPV1 receptor, capsazepine (50□M), when used, was preincubated for 3 minutes with cells in Ca²⁺ andMg²⁺-free HBSS followed by activation using the components as describedabove with 50 μM capsazepine. Cells were then fixed and were mountedonto glass slides.

GluR4 Receptor Activity Assay

HEK-293 cells were cotransfected with GluR4 (pRK5-GluR4, about 0.33μg/well), MLS-NR2B (pSGH-MLS-NR2B, about 0.33 μg/well) and GFP-CaMKII(pEGFP-CaMKII, about 0.1 μg/well) using lipofectamine reagent. Duringgrowth, the medium was supplemented with 50 μM CNQX (inhibitor of AMPAreceptor). After 48 hours, the transfected cells were subjected to GluR4activity assay. The growth medium was removed and the cells were washedtwice with Ca²⁺ and Mg free HBSS containing 2 mM HEPES and 400 μM EGTA.The GluR4 receptor was activated with HBSS solution containing 10 μMglutamate with or without 2 mM Ca²⁺ for about 5 minutes. The inhibitor,CNQX (50 μM), when used, was preincubated for 3 minutes with cells inCa²⁺ and Mg²⁺-free HBSS followed by activation using the components asdescribed above with 50 μM CNQX. Cells were then fixed and were mountedonto glass slides.

Basic System for Detection of Intracellular Ca²⁺-Release

The system described in FIG. 1 was imaged by confocal and non-confocalfluorescence microscopes and the images are presened in FIG. 2. Thearrows in the Plus-Ca²⁺ images indicate the cells which develop punctateappearance due to mitochondrial localization of GFP-α-CaMKII, consequentto intracellular rise in [Ca²⁺]. Quantification of cell count carriedout as described in FIG. 4 is shown in the confocal Plus-Ca²⁺ image.

Development of Ca²⁺-Dependent Signals by Using Full-Length NR2B Insteadof MLS-NR2B

The data in FIG. 3 is obtained by transfecting cells with full-lengthNR2B instead of MLS-NR2B. Since full length NR2B is localized to nuclearand other intracellular membranes, GFP-CaMKII localizes to thosemembraneous structures instead of mitochondria, upon sensing increase inintracellular [Ca²⁺].

Quantitation of the Intracellular Ca²⁺-Dependent Signal

The inventors have found that the number of cells that develop theGFP-clustering signal is dependent on the concentration of extracellularCa²⁺ and hence the intracellular Ca²⁺ (FIG. 6). It also depends on theefficiency of the binding between CaMKII and NR2B (FIG. 5). Theinventors developed a method to express quantitatively the strength ofthe signal as mentioned in Methods section (Methodology forTransfection, Ca²⁺-stimulation and imaging). Data in FIG. 4 is used todemonstrate this method.

In image1 of FIG. 4, there are totally 11 cells expressing GFP of whichtwo cells (numbered 3 and 7) show clustering/aggregation of GFP as seenby regions of intense fluorescence. Percentage of cells showingclustering is obtained as 2*100/11=27%.

In image 2, no cells are found to have the GFP clustering, out of the 6cells expressing GFP.

WT-CaMKII vs T286D-CaMKII—Cell Count Comparison

The cell count determined as mentioned in methods section as well as inthe previous section, for the wild-type and T286D mutant forms ofGFP-α-CaMKII are presented in FIG. 5. The T286D mutation which is knownto enhance the Ca²⁺/calmoduin affinity of α-CaMKII also improved itsefficiency of binding in the intracellular binding system as seen by theincreased (more than two fold) cell count (FIG. 5).

[Ca²⁺]-Dependence of Cell Count—WT and T286D-α-CaMKII

Different concentrations of extracellular Ca²⁺ are expected to produceproportional increases in intracellular [Ca²⁺] when ionomycin-mediatedCa²⁺-influx was generated. This was reflected in the cell count obtainedfor different extracellular [Ca²⁺] (FIG. 6). As [Ca²⁺] increased, thecell count also increased. Moreover the T286D mutant of GFP-α-CaMKIIwhich showed enhanced signal compared to GFP-(WT)-α-CaMKII (FIG. 5) alsoshowed improved sensitivity to [Ca²⁺] as seen by response at lower[Ca²⁺] upto 2 nM. This observation of dependence of the signal on [Ca²⁺]is the key advancement in our invention making this method suitable forsensing intracellular [Ca²⁺] in a semiquantitative manner.

Mutants of α-CaMKII and NR2B or NR2A that Generate a Localization Signalin Response to Intracellular Release of Ca²⁺

The following pairs of mutants of GFP-α-CaMKII and either of NR2A orNR2B sequences when expressed in HEK-293 cells exhibited. Ca²⁺-dependentbinding and generation of localization signals. Some of these pairs,generated signals more than the wild-type pair of GFP-α-CaMKII andMLS-NR2B. These could be used either as detection systems with improvedsensitivity or under cellular environments where the wild-type pairencounters technical problems such as faulty expression.

-   1. T286D-α-CaMKII and MLS-WT-NR2B-   2. F293E/N294D-α-CaMKII and MLS-WT-NR2B-   3. MLS-(S1303A)-NR2B and WT-α-CaMKII-   4. S1303A-NR2B and WT-α-CaMKII-   5. K42R/T286D-α-CaMKII and MLS-WT-NR2B-   6. MLS-Delta^(IN)NR2A (deletion of Ile¹²⁸⁶-Asn¹²⁸⁷ motif of NR2A)    and WT-α-CaMKII-   7. MLS-(S1291A)/Delta^(IN) (deletion of Ile¹²⁸⁶-Asn¹²⁸⁷ motif of    NR2A) NR2A and WT-α-CaMKII-   8. MLS-Delta^(IN)NR2A (deletion of Ile¹²⁸⁶-Asn¹²⁸⁷ motif of NR2A)    and T286D-α-CaMKII-   9. Delta^(IN)NR2A (deletion of Ile¹²⁸⁶-Asn¹²⁸⁷ motif of NR2A) and    T286D-α-CaMKII-   10. MLS-K1292A-NR2B and WT-α-CaMKII    Interaction of the Binding Site on Eag with GFP-CaMKII for Detection    of Intracellular Release of Ca²⁺

The voltage gated potassium channel of Drosophila (Eag) that islocalised in synapses as well as in axons also interacts at the T-siteof CaMKII (Sun, X. X., Hodge, J. J., Zhou, Y., Nguyen, M., and Griffith,L. C. (2004) J Biol Chem 279, 10206-10214). The Eag amino acid residues731-803 (or longer or shorter sequences) which comprise the CaMKIIbinding motif was cloned into the pSGH vector to obtain an MLS-Eagfusion that could be targeted to mitochondria. When wild-type orsuitable mutant forms of GFP-CaMKII coexpressed with wild-type orsuitable mutant forms of MLS-Eag were activated with increase inintracellular Ca²⁺, the GFP-CaMKII should translocate to mitochondriagiving rise to stable green fluorescent punctae as observed in case ofMLS-NR2B. FIG. 7 shows the Ca²⁺-dependent localization ofGFP-(WT)-CaMKII to MLS-Eag. This system could also be a Ca²⁺-sensorsimilar to the CaMKII-NR2B system.

Any peptide sequence which binds to the T-site of CaMKII similar to NR2Bcould be used as a binding partner for CaMKII in a Ca²⁺-sensing system.

NMDA Receptor Activity

The neuronal N-methyl-D-aspartate (NMDA)-type glutamate receptor is aglutamate activated Ca²⁺-channel that has NR1 and NR2B as subunits.

When activated with glutamate and glycine it caused influx of Ca²⁺ fromthe extracellular medium leading to translocation of GFP-□-CaMKII toNR2B that is localized in the nuclear membrane, endoplasmic reticulumand plasma membrane. This signal, shown by arrows in FIG. 8, was notformed in the absence of extracellular Ca²⁺. The signal generation wasblocked by the NMDA receptor specific inhibitors, AP-5 and MK-801,further establishing that the signal was specific for the activity ofNMDA receptor.

Activity of NMDA Receptor containing NR2A Subunit

Functional NMDAR can be formed by NR1 and NR2A subunit also instead ofthe NR1/NR2B combination. However, since the NR2A subunit does not bindCaMKII, a translocation signal consequent to influx of Ca²⁺ will not beobtained. By additionally transfecting the mitochondrially localizedMLS-NR2B sequence, it is possible to detect the activity of theNR2A-containing NMDAR also in a Ca²⁺-dependent manner. The arrows inFIG. 9 show mitochondrially translocated GFP-α-CaMKII.

Detection and Measurement of the Activity of Other Ca²⁺-Channels

Many other proteins with intracellular Ca²⁺-releasing activity such asCa²⁺-channel proteins (nicotinic acetyl choline receptor channel (GrandoS A, Horton R M, Mauro T M, Kist D A, Lee T X, Dahl M V., J Invest.Dermatol., 1996, 107, p 412-418), the vanilloid receptor 1 (TRPV1)(Witte D G, Cassar S C, Masters J N, Esbenshade T, and Hancock A A.2002, J Biomol Screen. 7, p 466-475), G-protein coupled receptors(Fujii, R., Hosoya, M., Fukusumi, S., Kawamata, Y., Habata, Y., Hinuma,S., Onda, H., Nishimura, O., and Fujino, M. 2000, J. Biol. Chem., 275, p21068-21074), etc. could be expressed in a heterologous cell culturesystem to generate specific Ca²⁺-channel activity. Activity of all suchproteins could also be detected if they are co-expressed with a suitablepair of wild-type or mutant forms of GFP-CaMKII and NR2B, NR2A, Eag orany other protein which binds to the T-site of CaMKII, as described inearlier sections.

Vanilloid Rececptor (TRPV1)

Vanilloid receptor is a Ca²⁺-channel that is activated by capsaicin, oneof the active principle of hot chilli peppers (Caterina et al., 1997,Nature, Vol. 389, p 816-824). We have found that the activity ofvanilloid receptor (TRPV1) could be detected by our invention forCa²⁺-sensing. HEK-293 cells cotransfected with TRPV1, GFP-CaMKII and MLSNR2B upon activation with capsaicin (3 μM or 10 nM), developed greenpunctae in a Ca²⁺-dependent manner as signal for the Ca²⁺-channelactivity of TRPV1 receptor. The signal generation was specific fortransfection by TRPV 1, activation by capsaicin and also for thepresence of Ca²⁺ in the extracellular medium. Signal generation due toactivation of TRPV1 could be inhibited by the TRPV1-specific inhibitorcapsazepine (FIG. 10). All these results show that the activity of TRPV1receptor is being detected by our invention.

Neuronal AMPA-Type Glutamate Receptor GluR4

The homomeric AMPA-type glutamate receptor formed of the GluR4 subunitcauses Ca²⁺-influx upon activation by glutamate (Cushing, et al., 1999,J. Neurosci. Methods. Vol. 90, p 33-36). Upon transfection of HEK-293cells with the GluR4 subunit along with GFP-CaMKII and MLS-NR2B, it waspossible to detect the Ca²⁺-channel activity of the receptor. Thereceptor was activated by treatment with glutamate which caused theformation of green punctae due to translocation of GFP-CaMKII tomitochondrially localized. MLS-NR2B. Generation of the signal could beblocked by the specific inhibitor of the receptor, CNQX (FIG. 11).

1. A simple end point assay for quantitation of transient intracellularcalcium with a set of pairs of proteins components having differentlevels of sensitivity to concentration of calcium, with broadapplicability to many Ca²⁺ channel proteins comprising: i) generation ofexpression constructs for the fusion proteins having the Ca²⁺/calmodulindependent protein kinase II (CaMKII) phosphorylation sites of NR2A orNR2B subunits of N-methyl-D-aspartate receptor (NMDAR) or the voltagegated potassium channel of Drosophila (Eag) or any protein sequencewhich binds to the T-site of CaMKII similar to NR2B, conjugated tomitochondrial localizing signal sequence, or mutants of these sequencesas described herein ii) generation of mammalian expression constructs ofα-CaMKII as a chimera with green fluorescent protein (GFP-α-CaMKII) orits mutants as described herein iii) subjecting the constructs or itsmutants to the step of site-directed mutagenesis iv) subjecting to thestep of transfection, Ca²⁺ stimulation, imaging and quantification ofthe number of cells with Ca²⁺ dependent signal, wherein NMDA receptoractivity assay, TRPVI receptor activity assay, GJuR4 receptor activityassay are performed to detect the activity of Ca²⁺ channel proteins. 2.The simple end point assay for quantitation of transient intracellularCa²⁺ as claimed in claim 1, wherein the cDNAs of NR2A, encoding aminoacid residues 1265-1301 or NR2B, encoding amino acid residues 1271-1311,or Eag, encoding amino acid residues 731-803, harboring thephosphorylation site for CaMKII (Ser¹²⁹¹, Ser¹³⁰³, Thr⁷⁸⁷ of NR2A, NR2Band Eag respectively are subcloned into PSGH vector.
 3. The simple endpoint assay for quantitation of transient intracellular Ca²⁺ as claimedin claim 1, wherein the sequences of NR2A and NR2B form parts of fusionproteins with a mitochondrial targeting signal sequence, a myc-epitopeand GST at the N-terminal end, MLS-NR2A, MLS-NR2B or MLS-Eag.
 4. Thesimple end point assay for quantitation of transient intracellular Ca²⁺as claimed in claim 1, wherein the N terminal GFP fusion of α-CaMKlI isgenerated by using forward primer having nucleotide sequence as in seqID No.1 and reverse primer having nucleotide sequence as in seq ID No.2.5. The simple end point assay for quantitation of transientintracellular Ca²⁺ as claimed in claim 1, wherein for transfection,Human Embryonic kidney HEK-293 cells were seeded and after about 24hours co-transfected with plasmid constructs as described herein.
 6. Thesimple end point assay for quantitation of transient intracellular Ca²⁺as claimed in claim 1, wherein a specific method of quantitation is usedby taking total number of green fluorescent cells which develop punctaeas a signal for intracellular calcium release and representing it aspercentage of the total number green fluorescent cells as describedherein.
 7. The simple end point assay for quantitation of transientintracellular Ca²⁺ as claimed in claim 1, wherein for expression of NMDAreceptor, the cells are co-transfected with plasmid constructs asdescribed herein.
 8. The simple end point assay for quantitation oftransient intracellular Ca²⁺ as claimed in claim 1, wherein for theexpression of TRPVI, HEK-293 cells are co-transfected with TRPV1 alongwith GFP-α-CaMKII and MLS-NR2B as described herein.
 9. The simple endpoint assay for quantitation of transient intracellular Ca²⁺ as claimedin claim 1, wherein for the expression of GluR4 receptor activity,HEK-293 cells are co-transfected with GluR4 along with GFP-α-CaMKII andMLS-NR2B as described herein.
 10. The simple end point assay forquantitation of transient intracellular Ca²⁺ as claimed in claim 1,wherein the activity of any protein with Ca²⁺ channel activity oractivity of intracellular Ca²⁺ release could be detected byco-expressing them with GFP-α-CaMKII and MLS-NR2B.
 11. The simple endpoint assay for quantitation of transient intracellular Ca²⁺ as claimedin claim 1, wherein real time monitoring of signals is not required fordetection of transient intracellular Ca²⁺.
 12. The simple end pointassay for quantitation of transient intracellular Ca²⁺ as claimed inclaim 1, wherein high throughput assays for any protein with Ca²⁺channel activity or activity of intracellular Ca²⁺ release could becarried out.